scholarly journals LuxS Promotes Biofilm Maturation and Persistence of Nontypeable Haemophilus influenzae In Vivo via Modulation of Lipooligosaccharides on the Bacterial Surface

2009 ◽  
Vol 77 (9) ◽  
pp. 4081-4091 ◽  
Author(s):  
Chelsie E. Armbruster ◽  
Wenzhou Hong ◽  
Bing Pang ◽  
Kristin E. Dew ◽  
Richard A. Juneau ◽  
...  
Keyword(s):  
Haemophilus Influenzae ◽  
Otitis Media ◽  
Opportunistic Infections ◽  
Bacterial Species ◽  
Enzyme Linked Immunosorbent Assay ◽  
Nontypeable Haemophilus Influenzae ◽  
Bacterial Persistence ◽  
Soluble Mediator

ABSTRACT Nontypeable Haemophilus influenzae (NTHI) is an extremely common airway commensal which can cause opportunistic infections that are usually localized to airway mucosal surfaces. During many of these infections, NTHI forms biofilm communities that promote persistence in vivo. For many bacterial species, density-dependent quorum-signaling networks can affect biofilm formation and/or maturation. Mutation of luxS, a determinant of the autoinducer 2 (AI-2) quorum signal pathway, increases NTHI virulence in the chinchilla model for otitis media infections. For example, bacterial counts in middle-ear fluids and the severity of the host inflammatory response were increased in luxS mutants compared with parental strains. As these phenotypes are consistent with those that we have observed for biofilm-defective NTHI mutants, we hypothesized that luxS may affect NTHI biofilms. A luxS mutant was generated using the well-characterized NTHI 86-028NP strain and tested to determine the effects of the mutation on biofilm phenotypes in vitro and bacterial persistence and disease severity during experimental otitis media. Quantitation of the biofilm structure by confocal microscopy and COMSTAT analysis revealed significantly reduced biomass for NTHI 86-028NP luxS biofilms, which was restored by a soluble mediator in NTHI 86-028NP supernatants. Analysis of lipooligosaccharide moieties using an enzyme-linked immunosorbent assay and immunoblotting showed decreased levels of biofilm-associated glycoforms in the NTHI 86-028NP luxS strain. Infection studies showed that NTHI 86-028NP luxS had a significant persistence defect in vivo during chronic otitis media infection. Based on these data, we concluded that a luxS-dependent soluble mediator modulates the composition of the NTHI lipooligosaccharides, resulting in effects on biofilm maturation and bacterial persistence in vivo.

scholarly journals Phosphorylcholine Decreases Early Inflammation and Promotes the Establishment of Stable Biofilm Communities of Nontypeable Haemophilus influenzae Strain 86-028NP in a Chinchilla Model of Otitis Media

2006 ◽  
Vol 75 (2) ◽  
pp. 958-965 ◽  
Author(s):  
Wenzhou Hong ◽  
Kevin Mason ◽  
Joseph Jurcisek ◽  
Laura Novotny ◽  
Lauren O. Bakaletz ◽  
...  
Keyword(s):  
Haemophilus Influenzae ◽  
Otitis Media ◽  
Fluorescent Antibody ◽  
Phase Variable ◽  
Nontypeable Haemophilus Influenzae ◽  
Bacterial Surface ◽  
Culture Cells ◽  
Antibody Staining

ABSTRACT Nontypeable Haemophilus influenzae (NTHi) is a leading causative agent of otitis media. Much of the inflammation occurring during NTHi disease is initiated by lipooligosaccharides (LOS) on the bacterial surface. Phosphorylcholine (PCho) is added to some LOS forms in a phase-variable manner, and these PCho+ variants predominate in vivo. Thus, we asked whether this modification confers some advantage during infection. Virulence of an otitis media isolate (NTHi strain 86-028NP) was compared with that of an isogenic PCho transferase (licD) mutant using a chinchilla (Chinchilla lanigera) model of otitis media. Animals infected with NTHi 86-028NP licD demonstrated increased early inflammation and a delayed increase in bacterial counts compared to animals infected with NTHi 86-028NP. LOS purified from chinchilla-passed NTHi 86-028NP had increased PCho content compared to LOS purified from the inoculum. Both strains were recovered from middle ear fluids as long as 14 days postinfection. Biofilms were macroscopically visible in the middle ears of euthanized animals infected with NTHi 86-028NP 7 days and 14 days postchallenge. Conversely, less dense biofilms were observed in animals infected with NTHi 86-028NP licD 7 days postinfection, and none of the animals infected with NTHi 86-028NP licD had a visible biofilm by 14 days. Fluorescent antibody staining revealed PCho+ variants within biofilms, similar to our prior results with tissue culture cells in vitro (S. L. West-Barnette, A. Rockel, and W. E. Swords, Infect. Immun. 74:1828-1836, 2006). Animals coinfected with equal proportions of both strains had equal persistence of each strain and somewhat greater severity of disease. We thus conclude that PCho promotes NTHi infection and persistence by reducing the host inflammatory response and by promoting formation of stable biofilm communities.

scholarly journals Nasal Delivery of a Commensal Pasteurellaceae Species Inhibits Nontypeable Haemophilus influenzae Colonization and Delays Onset of Otitis Media in Mice

2020 ◽  
Vol 88 (4) ◽  
Author(s):  
Caitlyn M. Granland ◽  
Naomi M. Scott ◽  
Jean-Francois Lauzon-Joset ◽  
Jeroen D. Langereis ◽  
Camilla de Gier ◽  
...  
Keyword(s):  
Haemophilus Influenzae ◽  
Otitis Media ◽  
Influenza A ◽  
Clinical Score ◽  
Nasal Delivery ◽  
Tissue Homogenate ◽  
Nontypeable Haemophilus Influenzae ◽  
Content Type

ABSTRACT Nasopharyngeal colonization with nontypeable Haemophilus influenzae (NTHi) is a prerequisite for developing NTHi-associated infections, including otitis media. Therapies that block NTHi colonization may prevent disease development. We previously demonstrated that Haemophilus haemolyticus, a closely related human commensal, can inhibit NTHi colonization and infection of human respiratory epithelium in vitro. We have now assessed whether Muribacter muris (a rodent commensal from the same family) can prevent NTHi colonization and disease in vivo using a murine NTHi otitis media model. Otitis media was modeled in BALB/c mice using coinfection with 1 × 104.5 PFU of influenza A virus MEM H3N2, followed by intranasal challenge with 5 × 107 CFU of NTHi R2866 Specr. Mice were pretreated or not with an intranasal inoculation of 5 × 107 CFU M. muris 24 h before coinfection. NTHi and M. muris viable counts and inflammatory mediators (gamma interferon [IFN-γ], interleukin-1β [IL-1β], IL-6, keratinocyte chemoattractant [KC], and IL-10) were measured in nasal washes and middle ear tissue homogenate. M. muris pretreatment decreased the median colonization density of NTHi from 6 × 105 CFU/ml to 9 × 103 CFU/ml (P = 0.0004). Only 1/12 M. muris-pretreated mice developed otitis media on day 5 compared to 8/15 mice with no pretreatment (8% versus 53%, P = 0.0192). Inflammation, clinical score, and weight loss were also lower in M. muris-pretreated mice. We have demonstrated that a single dose of a closely related commensal can delay onset of NTHi otitis media in vivo. Human challenge studies investigating prevention of NTHi colonization are warranted to reduce the global burden of otitis media and other NTHi diseases.

Nontypeable Haemophilus influenzae infection impedes Pseudomonas aeruginosa colonization and persistence in mouse respiratory tract

2021 ◽  
Author(s):  
Natalie Lindgren ◽  
Lea Novak ◽  
Benjamin C. Hunt ◽  
Melissa S. McDaniel ◽  
W. Edward Swords
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Respiratory Tract ◽  
Haemophilus Influenzae ◽  
Respiratory Infections ◽  
Bacterial Species ◽  
Young Patients ◽  
Nontypeable Haemophilus Influenzae ◽  
Potential Significance ◽  
And Diffusion

Patients with cystic fibrosis (CF) experience lifelong respiratory infections which are a significant cause of morbidity and mortality. These infections are polymicrobial in nature, and the predominant bacterial species undergo a predictable series of changes as patients age. Young patients have populations dominated by opportunists that are typically found within the microbiome of the human nasopharynx, such as nontypeable Haemophilus influenzae (NTHi); these are eventually supplanted and the population within the CF lung is later dominated by pathogens such as Pseudomonas aeruginosa ( Pa ). In this study, we investigated how initial colonization with NTHi impacts colonization and persistence of Pa in the respiratory tract. Analysis of polymicrobial biofilms in vitro by confocal microscopy revealed that NTHi promoted greater levels of Pa biofilm volume and diffusion. However, sequential respiratory infection of mice with NTHi followed by Pa resulted in significantly lower Pa as compared to infection with Pa alone. Coinfected mice also had reduced airway tissue damage and lower levels of inflammatory cytokines as compared with Pa infected mice. Similar results were observed after instillation of heat-inactivated NTHi bacteria or purified NTHi lipooligosaccharide (LOS) endotoxin prior to Pa introduction. Based on these results, we conclude that NTHi significantly reduces susceptibility to subsequent Pa infection, most likely due to priming of host innate immunity rather than a direct competitive interaction between species. These findings have potential significance with regard to therapeutic management of early life infections in patients with CF.

scholarly journals Role of Sialic Acid and Complex Carbohydrate Biosynthesis in Biofilm Formation by Nontypeable Haemophilus influenzae in the Chinchilla Middle Ear

2005 ◽  
Vol 73 (6) ◽  
pp. 3210-3218 ◽  
Author(s):  
Joseph Jurcisek ◽  
Laura Greiner ◽  
Hiroshi Watanabe ◽  
Anthony Zaleski ◽  
Michael A. Apicella ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Haemophilus Influenzae ◽  
Middle Ear ◽  
Biofilm Formation ◽  
Mammalian Host ◽  
Nontypeable Haemophilus Influenzae ◽  
Biofilm Production ◽  
Tract Infections

ABSTRACT Nontypeable Haemophilus influenzae (NTHI) is an important pathogen in respiratory tract infections, including otitis media (OM). NTHI forms biofilms in vitro as well as in the chinchilla middle ear, suggesting that biofilm formation in vivo might play an important role in the pathogenesis and chronicity of OM. We've previously shown that SiaA, SiaB, and WecA are involved in biofilm production by NTHI in vitro. To investigate whether these gene products were also involved in biofilm production in vivo, NTHI strain 2019 and five isogenic mutants with deletions in genes involved in carbohydrate biosynthesis were inoculated into the middle ears of chinchillas. The wild-type strain formed a large, well-organized, and viable biofilm; however, the wecA, lsgB, siaA, pgm, and siaB mutants were either unable to form biofilms or formed biofilms of markedly reduced mass, organization, and viability. Despite their compromised ability to form a biofilm in vivo, wecA, lsgB, and siaA mutants survived in the chinchilla, inducing culture-positive middle ear effusions, whereas pgm and siaB mutants were extremely sensitive to the bactericidal activity of chinchilla serum and thus did not survive. Lectin analysis indicated that sialic acid was an important component of the NTHI 2019 biofilm produced in vivo. Our data suggested that genes involved in carbohydrate biosynthesis and assembly play an important role in the ability of NTHI to form a biofilm in vivo. Collectively, we found that when modeled in a mammalian host, whereas biofilm formation was not essential for survivability of NTHI in vivo, lipooligosaccharide sialylation was indispensable.

scholarly journals Haemophilus parainfluenzae Strain ATCC 33392 Forms Biofilms In Vitro and during Experimental Otitis Media Infections

2017 ◽  
Vol 85 (9) ◽  
Author(s):  
Bing Pang ◽  
W. Edward Swords
Keyword(s):  
Otitis Media ◽  
Opportunistic Infections ◽  
Extracellular Dna ◽  
Neutrophil Extracellular Trap ◽  
Close Relative ◽  
Infection Model ◽  
Bacterial Persistence ◽  
Content Type ◽  
Haemophilus Parainfluenzae

ABSTRACT Haemophilus parainfluenzae is a nutritionally fastidious, Gram-negative bacterium with an oropharyngeal/nasopharyngeal carriage niche that is associated with a range of opportunistic infections, including infectious endocarditis and otitis media (OM). These infections are often chronic/recurrent in nature and typically involve bacterial persistence within biofilm communities that are highly resistant to host clearance. This study addresses the primary hypothesis that H. parainfluenzae forms biofilm communities that are important determinants of persistence in vivo. The results from in vitro biofilm studies confirmed that H. parainfluenzae formed biofilm communities within which the polymeric matrix was mainly composed of extracellular DNA and proteins. Using a chinchilla OM infection model, we demonstrated that H. parainfluenzae formed surface-associated biofilm communities containing bacterial and host components that included neutrophil extracellular trap (NET) structures and that the bacteria mainly persisted in these biofilm communities. We also used this model to examine the possible interaction between H. parainfluenzae and its close relative Haemophilus influenzae, which is also commonly carried within the same host environments and can cause OM. The results showed that coinfection with H. influenzae promoted clearance of H. parainfluenzae from biofilm communities during OM infection. The underlying mechanisms for bacterial persistence and biofilm formation by H. parainfluenzae and knowledge about the survival defects of H. parainfluenzae during coinfection with H. influenzae are topics for future work.

scholarly journals Multiple Consecutive Lavage Samplings Reveal Greater Burden of Disease and Provide Direct Access to the Nontypeable Haemophilus influenzae Biofilm in Experimental Otitis Media

2007 ◽  
Vol 75 (8) ◽  
pp. 4158-4172 ◽  
Author(s):  
Magali Leroy ◽  
Howard Cabral ◽  
Marisol Figueira ◽  
Valérie Bouchet ◽  
Heather Huot ◽  
...  
Keyword(s):  
Gene Expression ◽  
Haemophilus Influenzae ◽  
Otitis Media ◽  
Vaccine Development ◽  
Ex Vivo ◽  
Direct Analysis ◽  
Direct Access ◽  
Nontypeable Haemophilus Influenzae ◽  
Nasopharyngeal Colonization

ABSTRACT The typically recovered quantity of nontypeable Haemophilus influenzae (NTHi) bacteria in an ex vivo middle ear (ME) aspirate from the chinchilla model of experimental otitis media is insufficient for direct analysis of gene expression by microarray or of lipopolysaccharide glycoforms by mass spectrometry. This prompted us to investigate a strategy of multiple consecutive lavage samplings to increase ex vivo bacterial recovery. As multiple consecutive lavage samples significantly increased the total number of bacterial CFU collected during nasopharyngeal colonization or ME infection, this led us to evaluate whether bacteria sequentially acquired from consecutive lavages were similar. Comparative observation of complete ex vivo sample series by microscopy initially revealed ME inflammatory fluid consisting solely of planktonic-phase NTHi. In contrast, subsequent lavage samplings of the same infected ear revealed the existence of bacteria in two additional growth states, filamentous and biofilm encased. Gene expression analysis of such ex vivo samples was in accord with different bacterial growth phases in sequential lavage specimens. The existence of morphologically distinct NTHi subpopulations with varying levels of gene expression indicates that the pooling of specimens requires caution until methods for their separation are developed. This study based on multiple consecutive lavages is consistent with prior reports that NTHi forms a biofilm in vivo, describes the means to directly acquire ex vivo biofilm samples without sacrificing the animal, and has broad applicability for a study of mucosal infections. Moreover, this approach revealed that the actual burden of bacteria in experimental otitis media is significantly greater than was previously reported. Such findings may have direct implications for antibiotic treatment and vaccine development against NTHi.

scholarly journals VapC-1 of Nontypeable Haemophilus influenzae Is a Ribonuclease

2007 ◽  
Vol 189 (14) ◽  
pp. 5041-5048 ◽  
Author(s):  
Dayle A. Daines ◽  
Mack H. Wu ◽  
Sarah Y. Yuan
Keyword(s):  
Haemophilus Influenzae ◽  
Growth Inhibition ◽  
Gene Pair ◽  
Gene Families ◽  
Upper Respiratory Tract ◽  
Nontypeable Haemophilus Influenzae ◽  
E Coli ◽  
Conserved Gene

ABSTRACT Nontypeable Haemophilus influenzae (NTHi) organisms are obligate parasites of the human upper respiratory tract that can exist as commensals or pathogens. Toxin-antitoxin (TA) loci are highly conserved gene pairs that encode both a toxin and antitoxin moiety. Seven TA gene families have been identified to date, and NTHi carries two alleles of the vapBC family. Here, we have characterized the function of one of the NTHi alleles, vapBC-1. The gene pair is transcribed as an operon in two NTHi clinical isolates, and promoter fusions display an inverse relationship to culture density. The antitoxin VapB-1 forms homomultimers both in vitro and in vivo. The expression of the toxin VapC-1 conferred growth inhibition to an Escherichia coli expression strain and was successfully purified only when cloned in tandem with its cognate antitoxin. Using total RNA isolated from both E. coli and NTHi, we show for the first time that VapC-1 is an RNase that is active on free RNA but does not degrade DNA in vitro. Preincubation of the purified toxin and antitoxin together results in the formation of a protein complex that abrogates the activity of the toxin. We conclude that the NTHi vapBC-1 gene pair functions as a classical TA locus and that the induction of VapC-1 RNase activity leads to growth inhibition via the mechanism of mRNA cleavage.

scholarly journals NontypeableHaemophilus influenzaeInitiates Formation of Neutrophil Extracellular Traps

2010 ◽  
Vol 79 (1) ◽  
pp. 431-438 ◽  
Author(s):  
Richard A. Juneau ◽  
Bing Pang ◽  
Kristin E. D. Weimer ◽  
Chelsie E. Armbruster ◽  
W. Edward Swords
Keyword(s):  
Otitis Media ◽  
Bacterial Species ◽  
Outer Membrane Proteins ◽  
Neutrophil Extracellular Traps ◽  
Bacterial Surface ◽  
Persistent Infections ◽  
Extracellular Traps ◽  
Molecular Patterns

ABSTRACTNontypeableHaemophilus influenzae(NTHI) is a leading cause of otitis media infections, which are often chronic and/or recurrent in nature. NTHI and other bacterial species persistin vivowithin biofilms during otitis media and other persistent infections. These biofilms have a significant host component that includes neutrophil extracellular traps (NETs). These NETs do not mediate clearance of NTHI, which survives within NET structures by means of specific subpopulations of lipooligosaccharides on the bacterial surface that are determinants of biofilm formationin vitro. In this study, the ability of NTHI and NTHI components to initiate NET formation was examined using anin vitromodel system. Both viable and nonviable NTHI strains were shown to promote NET formation, as did preparations of bacterial DNA, outer membrane proteins, and lipooligosaccharide (endotoxin). However, only endotoxin from a parental strain of NTHI exhibited equivalent potency in NET formation to that of NTHI. Additional studies showed that NTHI entrapped within NET structures is resistant to both extracellular killing within NETs and phagocytic killing by incoming neutrophils, due to oligosaccharide moieties within the lipooligosaccharides. Thus, we concluded that NTHI elicits NET formation by means of multiple pathogen-associated molecular patterns (most notably endotoxin) and is highly resistant to killing within NET structures. These data support the conclusion that, for NTHI, formation of NET structures may be a persistence determinant by providing a niche within the middle-ear chamber.

scholarly journals Nontypeable Haemophilus influenzae infection impedes Pseduomonas aeruginosa colonization and persistence in mouse respiratory tract

2021 ◽  
Author(s):  
Natalie Lindgren ◽  
Lea Novak ◽  
Benjamin Carter Hunt ◽  
Melissa S. McDaniel ◽  
W. Edward Swords
Keyword(s):  
Respiratory Tract ◽  
Haemophilus Influenzae ◽  
Respiratory Infections ◽  
Bacterial Species ◽  
Young Patients ◽  
Nontypeable Haemophilus Influenzae ◽  
Immune Priming ◽  
And Diffusion ◽  
Sequential Infection

Patients with cystic fibrosis (CF) experience lifelong respiratory infections which are a significant cause of morbidity and mortality. These infections are polymicrobial in nature wherein the predominant bacterial species changes as patients age. Young patients have populations dominated by pathobiont such as nontypeable Haemophilus influenzae (NTHi), that are eventually supplanted by pathogens such as Pseudomonas aeruginosa (Pa), which are more typical of late-stage CF disease. In this study, we investigated how initial colonization with NTHi impacts colonization and persistence of Pa in the respiratory tract. Analysis of polymicrobial biofilms in vitro by confocal microscopy revealed that NTHi promoted greater levels of Pa biofilm volume and diffusion. However, sequential infection of mice with NTHi followed by Pa  showed significant reduction in Pa in the lungs as compared to infection with Pa alone. Coinfected mice also had reduced severity of airway tissue damage and lower levels of inflammatory cytokines as compared mice infected with Pa alone. Similar results were observed using heat-inactivated NTHi bacteria prior to Pa introduction. Based on these results, we conclude that NTHi can significantly reduce susceptibility to subsequent Pa infection, most likely due to immune priming rather than a direct competitive interaction between species.  These findings have potential significance with regard to therapeutic management of early life infections in patients with CF

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